3.3
Regulation of mitochondrial permeability transition by ER Ca2+
spikes during apoptosisThe high [Ca2+]m that follows IP 3 R/RyR spikes has recently been linked to
mitochondrial release of apoptogenic factors through the opening of the permeability
transition pore (PTP). The PTP is a high-conductance, nonselective ion channel that
spans the IMM and OMM at points where the two membranes are in contact
(Crompton, 1999). The voltage-dependent anion channel (VDAC), located in the
OMM, and the adenine nucleotide translocator (ANT), located in the IMM, are the
core constituents of the PTP. Other components include matrix-localized
cyclosporine D (the target of the PTP suppressor, cyclosporine A [CsA]), hexokinase
II, creatine kinase, and the benozodiazepine receptor. BAX and BAK have been
reported to bind and stimulate opening of the PTP, while antiapoptotic Bcl-2 and
Bcl-xL prevent its opening (reviewed in Zamzami and Kroemer, 2001). In vitro, BAX
can form pores with either VDAC or ANT in planar lipid bilayers, and Bcl-2 can
inhibit the formation of these pores (Brenner et al., 2000; Shimizu et al., 2000).
Opening of the PTP is observed during most forms of apoptosis and has been
proposed to encourage the release of apoptotic IMS proteins either indirectly by
inducing matrix swelling and rupture of the OMM, or directly by forming pores in
the OMM with proapoptotic Bcl-2 family members. Consistent with either model,
inhibitors of the PTP, such as CsA, have been shown to prevent the release of Cyt.c
during apoptosis, at least in some contexts (Zamzami and Kroemer, 2001). One area
of contention, however, is the extent to which sustained PTP opening reflects an
initiating as compared to an amplification event in different forms of apoptosis-
signaling pathways.
It has been known for many years that mitochondrial Ca2+ overload is a strong
inducer of PTP opening in isolated mitochondria (Crompton, 1999). Elegant studies
by Hajnoczky and coworkers have recently demonstrated that ER Ca2+ spikes can
facilitate PTP activation during apoptosis in vivo (Szalai et al., 1999). In intact or
permeabilized cells, high mitochondrial Ca2+ loads generated by IP 3 R stimulation or
exogenous Ca2+ spikes lead to weak PTP activation, characterized by small, transient
mitochondrial depolarizations that are reversible and that do not release apoptotic
proteins from the IMS. ‘PTP flickering’ may be a normal process in mitochondrial
physiology (Crompton, 1999; Duchen, 2000). However, if cells are briefly challenged
with apoptotic agents that act on mitochondria, such as C2 ceramide or staurosporin,
mitochondria become sensitized to IP 3 R-induced Ca2+ spikes, resulting in complete
mitochondrial depolarization and release of Cyt.c. Inhibition of the mitochondrial
Ca2+ uniporter with RuRed, or addition of the PTP inhibitor CsA, completely
prevented both loss of ∆m and Cyt.c release, suggesting that these events are
dependent on mitochondrial Ca2+ uptake followed by PTP opening. Intriguingly, if
the cells were subsequently washed to remove the apoptotic stimulus and the IP 3 -
inducing agonist (ATP), ∆m fully recovered, but the cells eventually underwent
apoptosis. These results suggest that during apoptosis IP 3 R-driven Ca2+ spikes may
THE ROLE OF THE ENDOPLASMIC RETICULUM IN APOPTOSIS 109